3β-Hydroxysteroid dehydrogenase for pregnenolone in ovaries of rabbits in mid-pregnancy was examined with histochemical and biochemical methods. Histochemical detection was carried out by Wattenberg's method (1958). In biochemical experiments, ³H-pregnenolone (10 mμ moles/1 ml) and pregnenolone (60 μg/ml) were incubated with tissue homogenates in phosphate buffer (0.1 M, pH 7.4) containing 1-3 mg DPN for 10 to 30 minutes at 37°C in air. Activity of 3β-ol-dehydrogenase was represented by mμ moles Δ⁴-3-ketosteroids formed per gram tissue per hour.
Although a rather high activity of 3β-ol-dehydrogenase was observed in the interstitial gland by the histochemical technique, low or almost negative activity was shown in the corpus luteum of the pregnant rabbit. However, activity of 3β-ol-dehydrogenase by biochemical method was 7,000-8,000 mμ moles progesterone formed per g tissue per hour in the corpus luteum and 29,000-23,200 mμ moles/g/h in the interstitial gland showing high activity of 3β-ol-dehydrogenase in both tissues. From the results obtained, it is suggested that a solubility of substrate steroids into tissue would be the important factor for the result obtained by the histochemical method.

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In our previous series of experiments it was confirmed that the pituitary prolactin could bring about histochemical and biochemical fat mobilisation (almost diffuse sudanophilia and increase of oleic and linoleic acid in quantity) as well as an elevation of the ratio of RNA-P/DNA-P in the crop gland besides distinct proliferation, while so-called prolactin-like substances in blood plasma and urine of lactating women had no such characteristic actions except slight proliferation.
In the present paper, uptake of H³-thimidine or H³-uridine of the crop gland was autoradiographically investigated instead of DNA-P or RNA-P respectively comparing the action of pituitary prolactin with that of prolactin-like substances.
Pituitary prolactin and prolactin-like substances accelerated the uptake of H³-thimidine in a similar degree, showing common proliferative action of these substances. On the other hand, H³-uridine uptake was recognized merely following treatment with pituitary prolactin, suggesting its secreting property, which prolactin-like substances could not have. It seemed to be probable that endogeneous pituitary prolactin might be metabolized in organism into semi-active prolactin-like substances.

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Inactivation of ACTH in human plasma in vitro was studied using a natural ACTH preparation. Studies were also performed in order to clarify the relationship between structure and stability in fresh human plasma of various synthetic ACTH preparations. ACTH activity after the incubation at 37°C over a period of 1, 2 and 4 hours was measured by a modification of the method of Guillemin et al. Plasma corticosterone content was determined by a fluorometric method.
1) USP Reference Standard ACTH, one of the crude natural ACTH preparations, was not inactivated even after 4 hours of incubation in fresh human plasma, but it was significantly inactivated during the same period in plasma stored overnight in a freezer. These results demonstrate that fresh human plasma degrades ACTH very slowly, while plasma stored in the frozen state degrades it very rapidly.
2) Inactivation of ACTH was markedly accelerated in streptokinase activated fresh human plasma. Furthermore, ACTH was also significantly inactivated in a solution of streptokinase activated human fibrinolysin (plasmin). An experiment was also under-taken to determine whether or not plasmin inhibitors have inhibitory effect on the inactivation of ACTH in stored plasma. 1-Cysteine inhibited the inactivation of ACTH in stored plasma, whereas ε-aminocaproic acid and trans-4-aminomethylcycrohexane carboxylic acid had no such effect. These results may suggest that plasmin may play a role in the inactivation of ACTH in human plasma.
3) A highly purified sheep ACTH (α_s-ACTH) and synthetic 39 amino acid ACTH (α_p^1-39-ACTH) were as stable as the crude preparation over a period of 4 hours of incubation in fresh human plasma. These results demonstrate that both natural and synthetic ACTH preparations have identical stability in fresh plasma.
4) However, synthetic preparations with 26, 19 and 18 amino acids (α_b^1-26-ACTH, α^1-19-ACTH and α^1-18-ACTH) were progressively less stable in that order, than was the whole molecule. It would appear, therefore, that the chain length of the C-terminal portion is an important factor in preventing ACTH from inactivation.
5) Two synthetic 18 amino acid ACTH amides (α^1-18NH₂-ACTH and α^1-18NH₂-Gly¹-ACTH) were significantly more stable than α^1-18-ACTH in fresh plasma. A synthetic 25 amino acid ACTH amide with unnatural amino acid sequence (α^1-25NH₂-d_ser¹ Nleu⁴-Val²⁵-ACTH) was more stable than α_b^1-26-ACTH. These results suggest that not only the chain length but also the structure of ACTH may influence the stability of ACTH.

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The distributions of ACTH to various organs of rats were studied at several time intervals after the intravenous injection of ¹³¹I-labeled ACTH, ³H-labeled ACTH and unlabeled native ACTH, respectively. A native ACTH was labeled with ¹³¹I by the method of Greenwood and Hunter. This ¹³¹I-labeled ACTH preparation had no steroidogenic activity. ³H-ACTH was prepared by exposing a native ACTH to ³H gas and by purifying it using gel filtration. This ³H-ACTH preparation retained about 20% of the steroidogenic activity. Radioactivity of ¹³¹I-ACTH in the samples was measured by a well type scintillation counter after being dissolved by heating in a solution of 30% of KOH. Radioactivity of ³H-ACTH in the samples was determined by the method of hyamine hydroxyde using the Tri-Carb liquid scintillation spectrometer. Bioassay was performed by a modification of the method of Lipscomb and Nelson after extracting the tissues using the glacial acetic acid method of Payne et al.
1) Shortly after the administration of ¹³¹I-ACTH, most of the radioactivity was detected in the kidney, with the peak value of 42% of the administered dose, 6 minutes after the injection. However, the distribution of radioactivity was very little in the adrenal gland throughout the time after the injection.
2) After the intravenous injection of ³H-ACTH, the TCA precipitable radioactivity was found most markedly in the kidney, followed by the liver, although it was only at the level of 0.3% of the administered dose in the adrenal gland.
3) In the levels of the subcellular fractions of the kidney and liver from rats 2 or 6 minutes after the injection of ³H-ACTH, the TCA precipitable radioactivity was found most predominantly in the mitochondrial fraction, followed by the microsomal fraction, and least in the supernatant fraction when compared at the same dry weight. In the supernatant fraction of the adrenal gland, however, there was almost the same amount of the TCA precipitable radioactivity as in the sediments.
4) Six minutes after the injection of 5 mU of USP Reference Standard ACTH in rats, the biological activity had recovered 13.3% of the administered dose only from the extract of the kidney, although it could not be detected in the extracts of the liver and the adrenal gland.
These results demonstrate that kidney is the main site for distribution of exogenous ACTH.

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Inactivation of ACTH by tissue slices of rats was studied in vitro in Krebs-Ringer bicarbonate buffer of pH 7.4 with gas phase of 95% O₂- 5% CO₂,. Experiments were also undertaken to clarify the presence of ACTH inactivating systems in the subcellular fractions of the rat tissues. After the incubation at 37°C for 30 or 60 minutes, ACTH activity recovered from the incubation medium was assayed by a modification of the method of Guillemin et al.
1) Native ACTH was markedly inactivated during 30 minutes by such tissue slices as the kidney, liver, adrenal, spleen and muscle, and moderately by slices of the lung. However, slices of the adipose tissue and brain did not inactivate ACTH under the same conditions. When kidney slices were boiled before the incubation, no inactivation was observed. Synthetic 24 amino acid ACTH (α^1-24-ACTH) was inactivated more rapidly than native ACTH by kidney slices, and also inactivated partially by adipose tissue slices. These results may suggest that the difference in ACTH inactivating ability of various tissues would not be qualitative, but quantitative, and also that synthetic short chain polypeptide is less stable than the whole molecule not only in plasma but also with tissue slices.
2) The effect of various proteinase inhibitors on the inactivation of ACTH by kidney slices was studied. Antiplasmin drugs such as trans-4-aminomethylcycrohexane carboxylic acid, e-aminocaproic acid hexylester, and 1-cysteine had no inhibitory effect at low molar concentrations, although they inhibited the inactivation at high molar concentrations. However, p-chloromercuribenzoate markedly prevented the inactivation of ACTH during a period of 60 minutes of the incubation. While 2, 4-dinitrophenol and puromycin, a specific inhibitor for protein biosynthesis, had no inhibitory effect. Streptokinase, an z c ivator of the plasmin system, did not accelerate the inactivation of ACTH by kidney slices. These results may suggest that the enzymatic degradation is responsible for the inactivation of ACTH by tissues.
3) ACTH was significantly inactivated by the supernatant fractions from the rat kidney and liver obtained by differential centrifugation, while the microsomal fraction from the rat adrenal gland had marked ACTH-inactivating ability. The inactivation of ACTH by the supernatant fraction appears to be the enzymatic degradation, since proteolytic enzymes are known to be present in this fraction. The inactivation of ACTH by the microsomal fraction, however, may be explained by the binding of ACTH to the adrenal cells.
In conclusion, it is suggested that the kidney is the most important organ in inactivat-ing ACTH in the body, since it is the main site for distribution of ACTH.

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Attempts were made in this study to find whether any influence by a disorder of the thyroid gland effects the process of pregnancy and delivery, and also the symptoms of hyperthyroidism by pregnancy. Therefore, the laboratory examinations for the thyroid function were done on pregnant women, with and without hyperthyroidism. Additionally, a few statistical observations were also made on women with hyperthyroidism at about the age of menarche, the menstrual cycle and the sterility rate. The time when the struma was revealed was listed and summarized in order to differentiate whether the pregnant woman had been suffering from hyperthyroidism before pregnancy or not. It was found that pregnancy scarcely provocated the hyperfunction of the thyroid gland. On the other hand, toxemia of pregnancy was found in seven pregnant women out of ten pregnant women with hyperthyroidism who had been recently observed at our clinic. And also it was found by a statistical research on 275 women with the thyroid disease that the spontaneous abortion and the premature birth occurred twice as often in pregnant women suffering from the thyroid disease after pregnancy as compared with those before pregnancy.
Measurements of basal metabolic rate (BMR), protein bound iodine (PBI), serum cholesterol, and I¹³¹-triiodothyronine resin sponge uptake (RSU) were undertaken to seek for the relationship between thyroid disease and pregnancy. The value of BMR was found to be generally increased in the later period of pregnancy, in both the normal pregnant and the pregnant women with hyperthyroidism. And a significant difference was found between them, though it had been considered that the BMR value was useless for a diagnosis of pregnancy with hyperthyroidism. The PBI and serum cholesterol were found to be useless. A lower value of RSU was observed in the normal pregnant as compared with in the non-pregnant women and the pregnant women with hyperthyroidism showed a significantly higher value than normal pregnancy. So, it appeared that RSU determination was a useful weapon as a laboratory examination on the thyroid function of the pregnant women.
In order to investigate on the lower value of RSU in pregnant women, the following studies were made : I¹³¹-labelled T₃ solution incubated with the serum specimen for one hour was separated by the oxoid paper electrophoresis. It was found that most of I¹³¹-T₃ incubated with serum and moved to alpha 1 and 2 globulin fractions, and albumin fractionrespectively. Furthermore, in order to reveal the localization and the binding capacity of I¹³¹-T₃ the gelfiltration on Sephadex-G 100 was used on the incubated serum specimen and the isolated I¹³¹-T₃. It has been known that I¹³¹-T₃, was found mostly in the albumin in the serum specimens of the normal woman, the pregnant woman, the woman with hyperthyroidism and the pregnant woman with hyperthyroidism. In the normal pregnant woman and the pregnant woman with hyperthyroidism, the portion of the maximum binding capacity was seen in the closer part of the globulin. Concerning the amount of protein in each fraction and the radioactivity of I¹³¹-T₃,, the albumin fraction in the pregnant women was observed to be more decreased than in the normal women, the increase of the albumin fraction and the decrease of the globulin fraction were found in the woman with hyperthyroidism. The main specific count (count per mg protein) was observed in the side of the albumin fraction in the normal woman, the woman with hyperthyroidism and the pregnant woman with hyperthyroidism. And the higher binding capacity was revealed in sequences of the pregnant woman with hyperthyroidism, the normal woman and the woman with hyperthyroidism. On the other hand,

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In the course of running an endocrine diagnostic laboratory, it became apparent that a number of interfering substances which originated from endogenous or exogenous sources were becoming more of a problem in the colorimetric procedures. Numerous attempts have been made to minimize these interferences while maintaining the speed and simplicity required for a routine clinical method.
In this investigation, the possibility of interference of medications clinically utilized in Porter-Silber and Zimmermann reaction was studied. The Porter-Silber chromogen was estimated by means of a modification of the method of Reddy, Jenkins, and Thorn, and the Zimmermann chromogen by means of a modification of Sobel's method.
1) About one hundred drugs, which are administrated to the patients frequently in our hospital, were studied concerning their reactivity in these reactions, because these drugs could be the exogenous interfering substances.
2) The pure substances of these medications were reacted in vitro to phenylhydrazinesulfuric acid and meta-dinitrobenzene, and the absorption spectra of the chromogen between 350 mμ and 600 mμ were examined. In the Porter-Silber reaction the extinction of chromogen at 410 mp could be accounted for as the source of interference, and the convexity or concavity of absorption spectra between 480 mμ and 560 mμ of Zimmermann chromogen, when Allen's formula was adapted, could interfere in this reaction. In this experiment, 36 of 100 drugs were suggested to be interfering sources. These medications were as follows :
For Porter-Silber reaction :
Triacetyloleandomycin, Colimycin, Cephalothin, Erythromycin, Nystatin, Glyseofulvin, Streptomycin, Ethionamide, Trichlormethiazide, Methychlothiazide, Hydroflumethiazide, Furosemide, Spironolactone, Oxyphenbutazone, N-methylcarbamate, Carbazochrome sodium sulfate, Ca-mesoxalate, Chlorpromazine, Dipyridamole, Digitoxin. For Zimmermann reaction :
Meprobamate, Chlordiazepoxide, Tranexamic acid, Oxyphenbutazone, Priscol, Ethionamide, Triamterene, Spironolactone, N-methylcarbamate, Acetylphenolphthalein, Isovalerylphenolphthalein.
3) The recovery of these substances to organic solvents which are used for the extraction of urinary corticosteroids was investigated by adding each pure substance to controlurine specimen. The influence of each added material was examined by estimating PorterSilber and Zimmermann chromogen of each specimen. Twelve of 36 substances were recovered and were revealed to be positive or negative interference in these reactions.
4) The drugs, which could exhibit the interference in the reactions from the results mentioned above, were investigated in vivo. The examined medications were Cephalothin, Colimycin, Ethionamide, Trichlormethiazide, Methychlothiazide, N-methylcarbamate, Glyseofulvin and Triacetyloleandomycin for Porter-Silber reaction and Meprobamate, Ethionamide and Spironolactone for Zimmermann reaction. Each drug was studied in three individuals who were healthy volunteers or patients who were hospitalized for long time without any endocrine disorders. For the first three days no drugs were administrated and the urine specimens of this period were collected as control, and for the next successive three days the drug, which would be tested, was administrated at the usual dose. After the discontinuity of the drug administration, urine was collected for three further days as the control period.
5) From these in vivo studies, Cephalothin, Colimycin, Ethionamide, and Trichlormethiazide interfered in Porter-Silber reaction, and Meprobamate and Spironolactone in Zimmermann reaction.
6) Colimycin revealed interesting results. While this drug, showing an absorption maximum at 410 mi2 in vitro, was suggested to be not extracted in organic solvent from the urine in recovery experiment, the administration actually resulted large positive error in the determination of urinary Porter-Silber chromogen.

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Hormonal control of glycogen metabolism has been studied by measuring the phosphorylase activity and glycogen content in rat liver and muscle after the administration or depletion of various hormones.

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In the previous paper, alternations of glycogen metabolism under various hormonal conditions were studied by the measurement of liver and muscle phosphorylase activities, and an evidence suggested that various hormones showed the direct and indirect effects on this enzyme activity. In this report the effects of fasting and feeding on a high carbohydrate or high lipid diet upon the phosphorylase activity are described and the mechanism producing the apparent changes are also discussed.

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A number of experiments on production of murine cervical and endometrial carcinoma in our laboratory have shown that the growth of carcinomas is accelerated under several conditions in which estrogenic action on the uterus persists.
Recent biological and biochemical investigations on the acting mechanism of steroidal hormone on their target organs have strongly suggested that the selective uptake of the hormones by the target organs is essential.
Since the development of experimentally induced murine carcinomas are influenced by estrogenic action, it must be examined whether the carcinoma tissue rakes in estrogenic substance selectively just as the mother tissue does.
The present communication deals with the uptake labeled estradiol-17β in the murine organs, particularly in the uterus and carcinomas induced in the uterus by the methylcholanthrene string method.
A dose of 10 or 20 μc of ³H-estradiol-17β was injected subcutaneously into normal and tumor bearing mice. Radioactivities in various tissues were measured by Tri-carb liquid scintillation spectrometer with different intervals following the injection of the radioactive estrogen. The radioactive substances in the tissues were fractioned by the column chromatography of the Amberlite IRC-50 and then examined by the fluorometry and spectrophotometry in order to identify their chemical compositions.
The ether soluble radioactivity, incorporated in normal uterine tissues, endometrial adenocarcinoma and cervical squamous cell carcinoma of the uterus was larger in amount than in the other tissues and remained for long period of time. The ether soluble radioactivity in muscle, blood and liver was small in amount and disappered within a short period of time. The incorporation of the water soluble radioactivity was notable only for the liver. The ether soluble radioactivity in uterus, uterine adenocarcinoma and uterine squamous cell carcinoma contained a large amount of ³H-estradiol-17β and a small amount of ³H-estrone. The ratio of ³H-estradio1-17β to ³H-estrone in these tissues is over 10 to 1. The muscle, blood and liver contained a comparativly larger amount of ³H-estrone in the ether soluble radioactivity, and the ratio of ³H-estradiol-17β to ³H-estrone in these tissues is below 5 to 1.
The presence of a significant difference in ³H-estradiol-17β incorporation between target organs and non-target organs has thus been revealed. The experimentally produced murine uterine carcinoma has shown the same incorporation pattern of ³H-estradiol-17β to that of their mother tissue, i.e. retain simillar biological characteristics in regard to the behaviour the estrogen.

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CF#1 uniform strain of mouse was first used for bioassay of FSH. With the augmentation of HCG, satisfactory results were obtained in the determination of small amounts of FSH. The animals kept at 23°±4°C were most suitable for the assay. With doses ranging from 0.4 to 10.0 μg of NIH-FSH-S₂, and with augmentation of 0.25 IU of HCG, a linear dose response curve was obtained in regard to the uterine weight. LH (NIH-LHS₇), less than 10 μg in amount, had no effect on the FSH dose response curve, provided the amount of FSH contained in the specimen was over 2μg.
ACTH (CORTOPHINE, Organon), LTH (PROLACTIN, Teikoku Zoki), TSH (THYROTROPIN, Armour), Growth Hormone (NIH-GH-B₉), Oxytocin (SYNTOCINON, Sandoz), and Vasopressin (PITRESSIN, Teikoku Zoki), given in varying amounts, failed to evoke any significant uterine response.
Clinical data obtained using this method were as follows : Average urinary FSH excretion in normal adult men (7 cases) was 278.5±89.33; post menopausal women (7 cases) 2825.7±373.40; castrated women (5 cases) 299.0±92.74, 3 days, 2629.4±506.57, 1 month, 3142.4±358.60, 1 year, and 2343.0±265.03, 2 years, respectively, after ovariectomy with values of 1822.0±316.83 prior to ovariectomy; children (10 cases) 13.5±3.26; normal adult women with regular menstrual period (7 cycles of 4 subjects) 350.3±59.14 on 3rd menstrual day, 60.3±8.22 on 14th day, 121.6±10.31 on 17th day, 101.0±11.43 on 20th day, 70.3±14.32 on 23th day, 159.7±34.28 on 26th day. (all values are expressed in μg Equivalent NIH-FSH-S₂ per day).

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